Atrial Natriuretic Peptide Precursor Research Articles

Corin: A Key Mediator in Sodium Homeostasis, Vascular Remodeling, and Heart Failure

Simple SummaryAtrial natriuretic peptide (ANP) is an important hormone that regulates many physiological and pathological processes, including electrolyte and body fluid balance, blood volume and pressure, cardiac channel activity and function, inflammatory response, lipid metabolism, and vascular remodeling. Corin is a transmembrane serine protease that activates ANP. Variants in the CORIN gene are associated with cardiovascular disease, including hypertension, cardiac hypertrophy, atrial fibrillation, heart failure, and preeclampsia. The current data indicate a key role of corin-mediated ANP production and signaling in the maintenance of cardiovascular homeostasis. In this review, we discuss the latest findings regarding the molecular and cellular mechanisms underlying the role of corin in sodium homeostasis, uterine spiral artery remodeling, and heart failure.Atrial natriuretic peptide (ANP) is a crucial element of the cardiac endocrine function that promotes natriuresis, diuresis, and vasodilation, thereby protecting normal blood pressure and cardiac function. Corin is a type II transmembrane serine protease that is highly expressed in the heart, where it converts the ANP precursor to mature ANP. Corin deficiency prevents ANP activation and causes hypertension and heart disease. In addition to the heart, corin is expressed in other tissues, including those of the kidney, skin, and uterus, where corin-mediated ANP production and signaling act locally to promote sodium excretion and vascular remodeling. These results indicate that corin and ANP function in many tissues via endocrine and autocrine mechanisms. In heart failure patients, impaired natriuretic peptide processing is a common pathological mechanism that contributes to sodium and body fluid retention. In this review, we discuss most recent findings regarding the role of corin in non-cardiac tissues, including the kidney and skin, in regulating sodium homeostasis and body fluid excretion. Moreover, we describe the molecular mechanisms underlying corin and ANP function in supporting orderly cellular events in uterine spiral artery remodeling. Finally, we assess the potential of corin-based approaches to enhance natriuretic peptide production and activity as a treatment of heart failure.

IL-6 induced enhanced clearance of proANP and ANP by insulin-degrading enzyme in T1DM mice.

Cardiovascular disease (CVD) is a prevalent cause of morbidity and mortality in type I diabetes mellitus (T1DM). However, the pathophysiological mechanisms underlying the relationship between CVD, CVD risk factors, and T1DM have not yet been sufficiently explored. Here, we report that insulin-degrading enzyme (IDE) effectively degrades the precursor of atrial natriuretic peptide (proANP) in HEK293T cells. The pro-inflammatory cytokine IL-6 elicited a significant dose-dependent increase in IDE protein expression. Inhibition of the ERK/MAPK signaling pathway with selumetinib abolished the IL-6-stimulated increase in IDE protein levels and decreased ANP secretion in H9C2 cells. Importantly, the T1DM mouse model displayed lower proANP in the heart and ANP in serum, due to increased IDE expression and activity. Our results suggest a novel role of IL-6 in ANP metabolism via IDE and provide possibilities for new potential therapeutic strategies for diabetes-related cardiovascular complications.

Function and regulation of corin in physiology and disease.

Atrial natriuretic peptide (ANP) is of major importance in the maintenance of electrolyte balance and normal blood pressure. Reduced plasma ANP levels are associated with the increased risk of cardiovascular disease. Corin is a type II transmembrane serine protease that converts the ANP precursor to mature ANP. Corin deficiency prevents ANP generation and alters electrolyte and body fluid homeostasis. Corin is synthesized as a zymogen that is proteolytically activated on the cell surface. Factors that disrupt corin folding, intracellular trafficking, cell surface expression, and zymogen activation are expected to impair corin function. To date, CORIN variants that reduce corin activity have been identified in hypertensive patients. In addition to the heart, corin expression has been detected in non-cardiac tissues, where corin and ANP participate in diverse physiological processes. In this review, we summarize the current knowledge in corin biosynthesis and post-translational modifications. We also discuss tissue-specific corin expression and function in physiology and disease.

Plasma pro-atrial natriuretic peptide to estimate fluid balance during open and robot-assisted esophagectomy: a prospective observational study

BackgroundIt remains debated how much fluid should be administered during surgery. The atrial natriuretic peptide precursor proANP is released by atrial distension and deviations in plasma proANP are reported associated with perioperative fluid balance. We hypothesized that plasma proANP would decrease when the central blood volume is compromised during the abdominal part of robot-assisted hybrid (RE) esophagectomy and that a positive fluid balance would be required to maintain plasma proANP.MethodsPatients undergoing RE (n = 25) or open (OE; n = 25) esophagectomy for gastroesophageal cancer were included consecutively in this prospective observational study. Plasma proANP was determined repetitively during esophagectomy to allow for distinction between the abdominal and thoracic part of the procedure. The RE group was 15° head up tilted during the abdominal procedure.ResultsThe blood loss was 250 (150–375) (RE) and 600 ml (390–855) (OE) (p = 0.01), but the two groups of patients were provided with a similar positive fluid balance: 1705 (1390–1983) vs. 1528 ml (1316–1834) (p = 0.4). However, plasma proANP decreased by 21% (p < 0.01) during the abdominal part of RE carried out during moderate head-up tilt, but only by 11% (p = 0.01) during OE where the patients were supine. Plasma proANP and fluid balance were correlated in the RE-group (r = 0.5 (0.073–0.840), p = 0.02) and tended to correlate in the OE group (r = 0.4 (−0.045–0.833), p = 0.08).ConclusionThe results support that plasma proANP decreases when the central blood volume is compromised and suggest that an about 2200 ml surplus administration of crystalloid is required to maintain plasma proANP during esophagectomy.Trial registrationClinicaltrials.gov (NCT02077673). Registered retrospectively February 12th 2014.

Three molecular forms of atrial natriuretic peptides: quantitative analysis and biological characterization.

Atrial natriuretic peptide (ANP) is primarily produced in the heart tissue and plays a pivotal role in maintaining cardiovascular homeostasis in endocrine and autocrine/paracrine systems and has clinical applications as a biomarker and a therapeutic agent for cardiac diseases. ANP is synthesized by atrial cardiomyocytes as a preprohormone that is processed by a signal peptidase and stored in secretory granules as a prohormone. Subsequent proteolytic processing of ANP by corin during the secretion process results in a bioactive form consisting of 28 amino acid residues. Mechanical stretch of the atrial wall and multiple humoral factors directly stimulates the transcription and secretion of ANP. Secreted ANP elicits natriuretic and diuretic effects via cyclic guanosine monophosphate produced through binding to the guanylyl cyclase-A/natriuretic peptide receptor-A. Circulating ANP is subjected to rapid clearance by a natriuretic peptide receptor-C-mediated mechanism and proteolytic degradation by neutral endopeptidase. In humans, ANP is present as three endogenous molecular forms: bioactive α-ANP, a homodimer of α-ANP designated as β-ANP, and an ANP precursor designated as proANP (also referred to as γ-ANP). The proANP and especially β-ANP, as minor forms in circulation, are notably increased in patients with cardiac diseases, suggesting the utility of monitoring the pathophysiological conditions that result in abnormal proANP processing that cannot be monitored by inactive N-terminal proANP-related fragments. Emerging plate-based sandwich immunoassays for individual quantitation of the three ANP forms enables evaluation of diagnostic implications and net ANP bioactivity. This new tool may provide further understanding in the pathophysiology of cardiac diseases. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

The expression of NPPA splice variants during mouse cardiac development.

Natriuretic peptide precursor-A (NPPA) is an early and specific marker for functional myocardium of the embryonic heart. NPPA gene encodes for a precursor of atrial natriuretic peptide (ANP). So far, three alternatively spliced variants have been reported for NPPA in human. In mouse, no alternatively spliced transcript of NPPA has been reported. In the current study, we investigated the expression of NPPA gene during cardiac differentiation of mouse adipose-tissue-derived stem cells (ADSCs) and embryonic stem (ES) cells. As revealed by reverse-transcription polymerase chain reaction analysis, 2-week-differentiated cells expressed some cardiac-specific makers, including ANP. Three additional intron-retained splice variants of NPPA were also detected during cardiac differentiation of the ADSCs and ES cells. In addition, we detected three intron-retained splice variants of NPPA in 8.5-day mouse embryonic heart. In the mature cardiomyocytes of 1-week-old mice, only the correctly spliced isoform of NPPA gene was expressed. Freshly isolated stromal vascular fraction also expressed one intron-retained isoform of NPPA gene. In conclusion, our findings have provided evidence for the expression of intron-retained splices of NPPA mRNA during the early stages of mouse cardiogenesis as well as in the mouse adipose tissue.

The Prognostic Value of Midregional Proatrial Natriuretic Peptide in Patients with Hemorrhagic Stroke

Background: Atrial natriuretic peptide (ANP) is a well-known prognostic marker of outcome and mortality in patients with cardiovascular disease. Midregional proatrial natriuretic peptide (MR-proANP) is a stable fragment of the ANP precursor hormone. As a prognostic marker after ischemic stroke, it reliably predicts poststroke mortality and functional outcome. This study aimed to analyze the prognostic value of MR-proANP in patients with hemorrhagic stroke, i.e. subarachnoid (SAH) and intracerebral hemorrhage (ICH). Methods: MR-proANP was analyzed in patients with spontaneous SAH or spontaneous ICH. All patients were prospectively randomized into two treatment arms: (1) a prophylactic normothermia group with a target core temperature 36.5°C using endovascular cooling, and (2) a control group with conventional stepwise predefined fever management using antipyretic medication and surface cooling. Blood samples were obtained on admission and on days 4 and 7. Measurement of MR-proANP was performed in serum using sandwich immunoassay. The primary endpoint was functional outcome [assessed by the Glasgow Outcome Score (GOS)] and the secondary endpoints were mortality within 180 days after hemorrhagic stroke and influence of temperature on MR-proANP. A favorable outcome was defined as GOS 4-5, and the patients were considered to have a poor outcome with a 180-day GOS score between 1 and 3. Results: Analysis of MR-proANP was performed in 24 patients with spontaneous SAH and 22 patients with spontaneous ICH. MR-proANP was elevated on days 4 and 7 as compared to baseline levels (p < 0.05 and p < 0.001, respectively). High MR-proANP levels (>120 pmol/l) were associated with increased mortality and poor outcome (after 180 days; p < 0.05, respectively). There was no significant difference regarding MR-proANP serum concentrations between the endovascular and the control groups. Conclusions: Increased levels of MR-proANP are independently associated with poor functional outcome and increased mortality after 180 days in patients with hemorrhagic stroke. Endovascular temperature control had no significant influence on MR-proANP levels.

Large-scale Identification of Endogenous Secretory Peptides Using Electron Transfer Dissociation Mass Spectrometry

Mass spectrometry-based unbiased analysis of the full complement of secretory peptides is expected to facilitate the identification of unknown biologically active peptides. However, tandem MS sequencing of endogenous peptides in their native form has proven difficult because they show size heterogeneity and contain multiple internal basic residues, the characteristics not found in peptide fragments produced by in vitro digestion. Endogenous peptides remain largely unexplored by electron transfer dissociation (ETD), despite its widespread use in bottom-up proteomics. We used ETD, in comparison to collision induced dissociation (CID), to identify endogenous peptides derived from secretory granules of a human endocrine cell line. For mass accuracy, both MS and tandem MS were analyzed on an Orbitrap. CID and ETD, performed in different LC-MS runs, resulted in the identification of 795 and 569 unique peptides (ranging from 1000 to 15000 Da), respectively, with an overlap of 397. Peptides larger than 3000 Da accounted for 54% in CID and 46% in ETD identifications. Although numerically outperformed by CID, ETD provided more extensive fragmentation, leading to the identification of peptides that are not reached by CID. This advantage was demonstrated in identifying a new antimicrobial peptide from neurosecretory protein VGF (non-acronymic), VGF[554-577]-NH2, or in differentiating nearly isobaric peptides (mass difference less than 2 ppm) that arise from alternatively spliced exons of the gastrin-releasing peptide gene. CID and ETD complemented each other to add to our knowledge of the proteolytic processing sites of proteins implicated in the regulated secretory pathway. An advantage of the use of both fragmentation methods was also noted in localization of phosphorylation sites. These findings point to the utility of ETD mass spectrometry in the global study of endogenous peptides, or peptidomics.

17β-Estradiol-induced interaction of ERα with NPPA regulates gene expression in cardiomyocytes

17β-Oestradiol (E2) and its receptors (ERα and ERβ) are important regulators of physiological and pathological processes in the cardiovascular system. ER act in concert with other regulatory factors mediating oestrogenic effects. However, the underlying mechanisms modulating ER transcriptional activity are not fully elucidated. To gain better understanding of E2-induced ERα action in the human heart, we aimed to identify and functionally analyse interaction partners of ERα. Using yeast two-hybrid assays with a human heart cDNA library, we identified atrial natriuretic peptide precursor A (NPPA), a well-known cardiac hypertrophy marker, as a novel ERα interaction partner interacting in an E2-dependent manner. Mutation analyses and immunofluorescence data indicated that the LXXLL motif within NPPA is necessary for its E2-induced interaction with ERα, its action as a co-repressor of ERα, and its translocation into the nucleus of human and rat cardiomyocytes. Expression analysis and chromatin immunoprecipitation assays in a human left ventricular cardiomyocyte cell line, AC16, showed that NPPA interacts with E2/ERα, suppressing the transcriptional activity of ERα on E2-target genes, such as NPPA, connexin43, αactinin-2, nuclear factor of activated T-cells, and collagens I and III. We characterize for the first time an E2-regulated interaction of NPPA with ERα in cardiomyocytes, that may be crucial in physiological and/or pathological cardiac processes, thereby representing a potential therapeutic target.

The quest for an atrium-specific biomarker

Dear Sir, With great interest we read the article by Maass et al. [1] who started a ‘quest for an atrium-specific biomarker’ using oligodeoxyribonucleotide (ODN) microarrays to identify differences in gene expression profile between atrial and ventricular myocardium. One of the ‘atrium-enriched genes’ is Nppa that has a ‘fold A/V microarray’ of 3.33 [1], whereas in hearts of healthy human adults, the concentration of Nppa transcripts, which encode preproANP (a precursor of atrial natriuretic peptide or ANP), is >100-fold higher in atrial than in ventricular myocardium [2, 3]. The explanation for this apparent discrepancy is that gene expression profiling was done with RNA extracted from neonatal rat atrial and ventricular cardiomyocytes. This particular choice of tissue is highly peculiar, assuming that the biomarker, once found, will be used in material originating from adult human patients. Foetal (and neonatal) rat (and human) atrial and ventricular myocardium possess roughly equal proANP concentrations [2, 3]. In adults, proANP is no longer found in healthy rat and human ventricular myocardium [2, 3] but reappears under pathological conditions such as heart failure and hypertension [3, 4]. Furthermore, the heart of newborns changes from a mainly glucose-metabolising tissue to a primarily fatty acid-oxidising tissue, thereby upregulating the expression of genes involved in fatty acid transport and β-oxidation [5]. Another cautionary note concerns the tendency to equate transcript/RNA levels as determined by ODN microarray analysis with protein levels. This may not be justified because: (1) Many genetic loci specify all kinds of non-protein-coding transcripts besides the classical polyadenylated protein-coding mRNAs that might contribute to the signals observed on ‘DNA chips’ [6]; (2) Depending on the stringency of the hybridisation and washing conditions, the same DNA probe may be bound by related nucleic acid sequences with different coding capacity; and (3) Cells possess multiple mechanisms to control gene expression at the post-transcriptional level [7], many of which affect the ratio between a specific mRNA and the protein(s) it encodes. In conclusion, to find heart chamber type-specific proteins that will potentially allow the identification of atrium- or ventricle-specific biomarkers in human adults, the use of adult human atrial and ventricular RNA for gene expression profiling is recommended, provided any novel candidate is tested at the protein level, e.g. by Western blot analysis.

The genetics of loop diuretic effects

Little is known about the genetic impact on loop diuretic effects. We newly investigated five genetic polymorphisms in 95 healthy volunteers, who had ingested bumetanide, frusemide and torsemide. The subjects excreted means of 20.2 g sodium chloride, 2.87 g potassium and 261 mg calcium over 24 h. Concerning sodium chloride, the subjects excreted 2.2 g less per two T-alleles of C825T in the G nucleotide β-subunit 3 (GNB3), 3.2 g less per two Met32-alleles of Val32Met in the atrial natriuretic peptide precursor (ANP) and 2.8 g more per two Arg152-alleles of Ter152Arg in ANP (P=0.007, 0.05 and 0.007). Concerning potassium, the subjects excreted 0.42 g more per two ANP Arg152-alleles (P=0.023). Concerning calcium, the subjects excreted 32 mg more per two deletion-alleles of the insertion/deletion polymorphism in the angiotensin-converting enzyme, 44 mg more per two Trp460-alleles of Gly460Trp in α-adducin (ADD1) and 42 mg less per two GNB3 T-alleles (P=0.006, 0.023 and 0.008). The common genetic impact together with three polymorphisms in the sodium chloride cotransporter and the epithelial sodium channel was 20, 15, 10 and 23% of the variation in the urinary excretion of sodium chloride, volume, potassium and calcium. This exceeded the fraction of variation explained by differences in the pharmacokinetics: 13, 10, 11 and 6%. Thus, genetic variation seems to be a stronger predictor of the loop diuretic drug response than pharmacokinetic variation.

043 Admission MR-PROANP levels are superior to grace score and NT-PROBNP for risk stratification in non ST-elevation acute coronary syndromes: Abstract 43 Table 1

<h3>Introduction</h3> Endorsed by international guidelines, risk stratification plays a central role in the optimal management of patients with non ST-elevation (NSTE-ACS). High risk patients benefit from an early invasive approach and admission biomarker levels may help to identify this cohort. Atrial natriuretic peptide (ANP) is a recognised prognostic marker in ST elevation myocardial infarction (STEMI) and in heart failure (HF) but has not been evaluated in NSTE-ACS. The precursor of ANP is N-terminal Pro-ANP whose mid regional epitope is more stable when compared to epitopes of N- or C- terminals of pro-ANP used in previous assays. <h3>Aims</h3> To assess the prognostic value, optimum time for sampling and a cut off value for mid regional proANP in unselected patients9 with non ST elevation ACS (NSTE-ACS). We use GRACE risk score and NTproBNP as benchmark comparators. <h3>Method and Results</h3> Plasma MRproANP was measured both on admission (taken within 36 h of symptoms) and discharge (3 to 5 days later) in 630 (437 men, median age 70.0±12.8 years) patients with NSTE-ACS. During follow up which ranged from 150 to 1059 days the primary end point of a major adverse cardiac event (MACE) was reached by 170 (27%) patients. This included 84 (13.3%) deaths, 56 (8.9%) readmissions with HF and 68 (10.8%) re-infarctions. Admission and discharge levels were entered into 2 separate multivariate Cox regression models adjusted for important clinical and biochemical factors and Receiver Operator Characteristic (ROC) analyses performed with results shown in Abstract 43 Table 1. Admission levels demonstrated a stronger association with MACE were therefore used for comparison against GRACE score and admission NTproBNP level in a further multivariate Cox model. This showed that MRproANP (HR 5.81 p&lt;0.001) and GRACE score calculated pre-discharge (HR 1.01 p=0.024) continued to be independently associated with the primary end point but not admission NTproBNP (HR 1.00 p=0.99) or GRACE score if calculated on presentation (HR 1.01 p=0.057). The accuracy of MRproANP at prediction of the MACE (AUC 0.76 p&lt;0.001) was not improved by combining with GRACE score (AUC 0.72 p&lt;0.001) both (AUC 0.76 p&lt;0.001). ROC analyses provided the optimum cut off value for MRproANP for prediction of MACE of 165 pmol/L which gave optimum survival stratification on Kaplan–Meier analysis (log rank 90.12 p&lt;0.001). <h3>Conclusion</h3> MRproANP level is a potent prognostic marker of adverse events. Admission levels offer early identification of high risk NSTE-ACS patients and its role in optimising management requires further investigation.

Systemic molecular and cellular changes induced in rats upon inhalation of JP-8 petroleum fuel vapor

Limited information is available regarding systemic changes in mammals associated with exposures to petroleum/hydrocarbon fuels. In this study, systemic toxicity of JP-8 jet fuel was observed in a rat inhalation model at different JP-8 fuel vapor concentrations (250, 500, or 1000 mg/m3, for 91 days). Gel electrophoresis and mass spectrometry sequencing identified the α-2 microglobulin protein to be elevated in rat kidney in a JP-8 dose-dependent manner. Western blot analysis of kidney and lung tissue extracts revealed JP-8 dependent elevation of inducible heat shock protein 70 (HSP70). Tissue changes were observed histologically (hematoxylin and eosin staining) in liver, kidney, lung, bone marrow, and heart, and more prevalently at medium or high JP-8 vapor phase exposures (500–1000 mg/m3) than at low vapor phase exposure (250 mg/m3) or non-JP-8 controls. JP-8 fuel-induced liver alterations included dilated sinusoids, cytoplasmic clumping, and fat cell deposition. Changes to the kidneys included reduced numbers of nuclei, and cytoplasmic dumping in the lumen of proximal convoluted tubules. JP-8 dependent lung alterations were edema and dilated alveolar capillaries, which allowed clumping of red blood cells (RBCs). Changes in the bone marrow in response to JP-8 included reduction of fat cells and fat globules, and cellular proliferation (RBCs, white blood cells-WBCs, and megakaryocytes). Heart tissue from JP-8 exposed animals contained increased numbers of inflammatory and fibroblast cells, as well as myofibril scarring. cDNA array analysis of heart tissue revealed a JP-8 dependent increase in atrial natriuretic peptide precursor mRNA and a decrease in voltage-gated potassium (K+) ion channel mRNA.

Detection of Left Ventricular Hypertrophy in Rats Administered a Peroxisome Proliferator–Activated Receptor α/γ Dual Agonist Using Natriuretic Peptides and Imaging

Chronic treatment with suprapharmacologic doses of peroxisome proliferator-activated receptor (PPAR) agonists has a known potential for causing left ventricular hypertrophy (LVH). The mechanism by which LVH develops is not well understood nor are biomarkers of it well characterized. Natriuretic peptides are important regulators of cardiac growth, blood volume, and arterial pressure and may be useful biomarkers of LVH and hemodynamic changes that precede it. We measured amino-terminal pro-atrial natriuretic peptide (NTproANP), amino-terminal pro-brain natriuretic peptide (NTproBNP), and cardiac troponin I (cTnI) concentrations in serum and plasma, as well as transcripts in left ventricular heart tissue for atrial natriuretic peptide precursor (Nppa), brain natriuretic peptide precursor (Nppb), and myosin heavy chain-beta (Myh7) as potential biomarkers of LVH induced by a PPARalpha/gamma dual agonist in Sprague-Dawley rats. We used magnetic resonance imaging, echocardiography, and hemodynamics to identify structural and functional cardiovascular changes related to the biomarkers. Heart-to-brain weight ratios (HW:BrW) were correlated with NTproANP, NTproBNP, and cTnI concentrations in serum as well as fold change in expression of Nppa and Nppb. LVH was characterized by increased left ventricular wall thickness and inner diameter, increased cardiac output, decreased arterial blood pressure, and increased heart rate. In these studies, each end point contributed to the early detection of LVH, the ability to monitor its progression, and demonstrated the ability of NTproANP concentration in serum to predict LVH and hemodynamic changes.

Epigenetic regulation and gene expression of vasopressin and atrial natriuretic peptide in alcohol withdrawal

Disturbances of volume regulating peptides like vasopressin and atrial natriuretic peptide (ANP) have been described in early abstinent patients. Aim of the present study was to evaluate possible alterations of the promoter-related DNA methylation of the ANP and vasopressin precursor genes and the related mRNA-expression of these genes in early alcohol withdrawal. We analyzed blood samples of 57 healthy controls and of 111 patients suffering from alcohol dependence that were admitted for detoxification treatment. Promoter-related DNA methylation and mRNA-expression of vasopressin and ANP genes were assessed using real-time PCR. Vasopressin mRNA-expression was not statistically different between patients and controls. However, we found a significantly elevated promoter-related DNA methylation of the vasopressin gene in patients with alcohol dependence (Mann-Whitney U-test: Z=-2.178, p=0.029). ANP mRNA-expression was significantly elevated in alcoholic patients (Z=-6.240, p<0.001) while promoter-related DNA methylation of ANP was significantly decreased (Z=-2.282, p=0.023). Furthermore, promoter-related DNA methylation of ANP was significantly correlated to the extent of craving measured with the OCDS (r=-0.197, p=0.040). The findings of the present study show significant alterations of the mRNA-expression and promoter-related DNA methylation of vasopressin and especially ANP precursor genes in patients with alcohol dependence. Further studies focusing on longitudinal changes of epigenetic regulation and gene expression of both peptides are needed to clarify the pathophysiological role of these findings.

Abstract 1477: Identification and Functional Analysis of Protein Interaction Partners of the Estrogen Receptor Alpha in the Human Heart

Background and aims: Estrogens (E2) are important regulators of physiological and pathological processes in various target tissues, including the human heart. Most of the biological actions of E2 are mediated by nuclear steroid receptors, the estrogen receptor alpha (ERα) and beta (ERα), which function as hormone-inducible transcription factors. ERs act in concert with other regulatory elements to mediate estrogenic effects. So far, only few cofactors of ERs have been described in the human heart. To gain a better understanding of E2-mediated ERα action in the human heart, we identified and functionally analyzed the interaction partners of ERα. Methods: The yeast two hybrid screen (YTH) was performed in the presence of E2 with ERα as bait in a human heart cDNA library. Co-immunoprecipitation analysis (Co-IP) with human atrial proteins was carried out to confirm the interaction in a mammalian system. Immunofluorescence (IF) methods were employed on human atrial tissue samples as well as in cotransfected AC16 cells to analyze the co-localisation of ERα and its binding partners. Promoter reporter assays were performed to show the regulation of the identified interaction partner by ERα. Results: Yeast two hybrid screening of a human heart library revealed that ERα interacts with the atrial natriuretic peptide precursor A (NPPA). Retransformation experiments showed that NPPA can interact with ERα full-length and the ERα-EF domain in an E2-dependent manner. The LxxLL-motif of NPPA was found to interact with the ERα-EF domain in yeast. The interaction of NPPA and ERα was also confirmed using proteins derived from human atrium by Co-IP. IF analysis showed that E2 treatment of AC16 cells led to an increased translocation of NPPA and ERα in the nucleus of AC16 cells and also their co-localization there. Furthermore, expression analysis with NPPA luciferase promoter constructs showed a significant increase in the promoter activity in the presence of E2/ERα. Conclusion: We showed the interaction of ERα and NPPA in an E2-dependent manner. Our findings suggest that the E2-activated ERα and NPPA complex binds to specific regions on the NPPA promoter leading to transcriptional regulation of the NPPA gene.

Selected News Items

1.Morita H, Rehm HL, Menesses A, McDonough B, Roberts AE, Kucherlapati R, Towbin JA, Seidman JG, Seidman CE. Shared genetic causes of cardiac hypertrophy in children and adults. N Engl J Med . 2008;358:1899–1908. PMID: 18403758. ### Study Hypothesis Mutations in sarcomere proteins that are implicated in adult forms of hypertrophic cardiomyopathy may play a role in idiopathic cardiac hypertrophy with onset in childhood, a disorder associated with poor prognosis. ### How Was the Hypothesis Tested? Morita and colleagues1 screened for mutations in 8 genes encoding sarcomeric proteins (MYH7, MYBPC3, TNNT2, TNNI3, TPM1, MYL3, MYL2, and ACTC) using direct sequencing techniques in 84 children with idiopathic cardiac hypertrophy. Mutations in these genes cause adult-onset cardiomyopathy. ### Principal Findings The authors identified mutations in almost half of children without a positive family history of cardiomyopathy (25 of 51 affected children) and in two thirds (21 of 33) of affected children with familial cardiomyopathy. Mutations in MYH7 (encoding β-myosin heavy chain) and MYBPC3 (encoding myosin-binding protein C) were the most frequent variants identified in the children. In the subgroup with sporadic (nonfamilial) cardiac hypertrophy, boys were more likely than girls to harbor mutations. ### Implications The authors conclude that childhood onset of cardiac hypertrophy should provoke genetic investigation and family assessment because about 50% of cases in this series were attributable to mutations routinely screened for in adults with unexplained cardiac hypertrophy. Identification of such mutations in children with cardiac hypertrophy would raise the possibility that management strategies used for adults with genetic cardiac hypertrophy may improve clinical outcomes in these children. 2.Lynch AI, Boerwinkle E, Davis BR, Ford CE, Eckfeldt JH, Leiendecker-Foster C, Arnett DK. Pharmacogenetic association of the NPPA T2238C genetic variant with cardiovascular disease outcomes in patients with hypertension. JAMA . 2008;299:296–307. PMID: 18212314. ### Study Hypothesis Variation in NPPA gene, which encodes the precursor of atrial natriuretic peptide, may influence the efficacy of different …

Apoptosis Signal-Regulating Kinase 1 Attenuates Atrial Natriuretic Peptide Secretion

Atrial natriuretic peptide (ANP) is an endogenous peptide hormone that is synthesized and secreted by the myocardium in health and disease. Although the bioactivity of this molecule has been studied extensively, cellular mechanisms governing its processing and secretion are not fully understood. Through a yeast two-hybrid screen of a cDNA library made from tissue of a failing human heart, we have discovered that the precursor of ANP, natriuretic peptide precursor (NPPA), physically interacts with the N-terminus of apoptosis signal-regulating kinase 1 (ASK1), a kinase believed to be involved in the pathogenesis of heart failure. We demonstrated that NPPA is a substrate of ASK1 in an in vitro kinase assay. Indirect immunofluorescence microscopy shows that, when expressed in Hela cells, ASK1 and NPPA exhibit distinct, but overlapping, staining patterns, suggesting partial colocalization in cells. Additionally, coexpressing wild-type ASK1 with NPPA in Hela cells led to reduced levels of NPPA in the culture medium, suggesting that ASK1 negatively impacts NPPA processing and/or secretion. This negative effect was less pronounced when a dominant-negative allele of ASK1 with deficient kinase activity was coexpressed with NPPA. Because both ASK1 and ANP are associated with pathologic cardiac hypertrophy, their interaction may have pathophysiological and therapeutic relevance.

Epigenetic Downregulation of Atrial Natriuretic Peptide but not Vasopressin mRNA Expression in Females with Eating Disorders is Related to Impulsivity

Disturbances of volume-regulating mechanisms have already been implicated in the pathophysiology of eating disorders like anorexia or bulimia nervosa with the peptide hormones vasopressin and atrial natriuretic peptide (ANP) being of special interest. Aim of the present study was to investigate, whether the expression of the corresponding genes was altered and if so, if these changes could be explained by epigenetic mechanisms such as DNA methylation. We analyzed blood samples of 46 women suffering from anorexia (n=22) or bulimia nervosa (n=24) as well as of 30 healthy controls. Peripheral mRNA expression and DNA methylation of the vasopressin and the ANP precursor genes were assessed using real-time PCR. We found significantly lower levels of ANP mRNA in patients with eating disorders. This downregulation was accompanied by a hypermethylation of the ANP gene promoter in the bulimic subgroup. We did not find differences regarding expression or methylation of the vasopressin gene. ANP mRNA expression was inversely associated with impaired impulse regulation. We conclude that epigenetic mechanisms may contribute to the known alterations of ANP homeostasis in women with eating disorders.

Nitric Oxide Induces Gene Expression of Jumonji and Retinoblastoma 2 Protein while Reducing Expression of Atrial Natriuretic Peptide Precursor Type B in Cardiomyocytes

Nitric Oxide Induces Gene Expression of Jumonji and Retinoblastoma 2 Protein while Reducing Expression of Atrial Natriuretic Peptide Precursor Type B in Cardiomyocytes